Diamond has many appealing properties, including biocompatibility, ease of surface modification, and chemical–physical stability. In this study, the temperature dependence of the pH-sensitivity of a oxygen-terminated boron-doped diamond solution gate FET (C-O BDD SGFET) is reported. The C-O BDD SGFET operated in an electrolyte solution at 95 °C. At 80 °C, the pH sensitivity of C-O BDD SGFET dropped to 4.27 mV/pH. As a result, we succeeded in developing a highly sensitive pH sensing system at −54.6 mV/pH at 80 °C by combining it with a highly pH sensitive stainless-steel vessel. It means that the thickness and volumetric concentration of boron is quite crucial to obtain FET characteristics.Measuring pH is required in a variety of industries, including the food manufacturing, medical, and agriculture industries. Various types of pH sensors have been developed to meet these needs. The most widely used of these are glass electrode pH sensors, because of their long-term stability and good reproducibility. However, conventional glass electrode pH sensors have the risks of leakage of the KCl internal solution or breakage of the glass electrode. To prevent these risks, glass electrode pH sensors have not been applied in the food industry, and all-solid-state pH sensing systems have received a great deal of attention. In the 1970s, ion-sensitive field-effect-transistors (ISFETs), which can act as a biosensor in the solution, were reported from Piet Bergveld . Much reDiamond SGFETs are also good candidates for pH-sensing applications, since the hole concentration of boron-doped diamond SGFETs varies with different ion concentrations of the solution . These tThe temperature characteristics of silicon ISFETs remain difficult, and the temperature characteristics of other semiconductor pH sensors have only been partially studied. AlGaN/GaN heterostructure pH sensors can operate at temperatures as high as 80 °C, and their pH sensitivity has been found to be stable and highly sensitive at room temperature . The AlG2 were used in this study [DS-VGS, and the other to measure the temperature dependence of pH sensitivity. Both substrates were made in the same way. Boron-doped layer was deposited on the polycrystalline diamond using a microwave plasma chemical vapor deposition (CVD) . The CVD conditions named in the previous paper were used [3) and sulfuric acid (H2SO4) at 200 °C for 30 min before the surface of polycrystalline diamond substrates was fully hydrogen-terminated (C-H) with microwave-enhanced plasma CVD (Astex: AX5010). Then, gold source and drain electrodes were deposited on the C-H diamond surface, except for the channel region, by using electron-beam evaporation with a thickness of 100 nm. The channel length L and width W were 200 μm and 5 mm. To connect the wires to the gold electrodes, conductive epoxy resin was used, and then all the conductive parts were covered with insulating epoxy resin. Oxygen termination (C-O) on the channel region of the diamond surface was carried out by a plasma reactor in an oxygen atmosphere. The cross-sectional diagram of C-O BDD SGFET is shown in Two black polycrystalline diamond substrates with dimensions of 5 × 5 mmis study ,24. One ere used . The surGS with the same drain current from the measurement results of the IDS-VGS characteristic.The pH sensitivity was measured in the Carmody buffer solutions prepared in the same way as in the previous paper . The pH DS drain-source voltage VDS characteristics at room temperature, 60, 80, and 95 °C when using an Ag/AgCl electrode and vessel gate, respectively. As the temperature increased, the drain current increased and the transconductance increased, and the gate leakage current increased at low drain-source voltage. The transconductance levels were 11, 13, 24, 34 μS for e and pμ is elementary charge and hall mobility, AN is accepter concentration, Z is gate width, and h is boron-doped depth. The transconductance of BDD SGFET is related to channel conductance in junction FET, however, this formula assumes that the acceptor is completely ionized [AN is replaced with hole concentration p expressed asDN is the compensating donor concentration, vM is the effective density of states of the valence band, dg is the acceptor degeneracy, iE is the ionization energy of the acceptor, Bk is the Boltzmann constant, and T is the temperature [p depends on the exponential term, which has a temperature parameter, and hence the channel conductance of BDD SGFET increases with increasing temperature. Here, since the channel conductance was tripled at 95 °C, it can be expected that the temperature-dependent exponential term was tripled compared to that at room temperature. The corresponding activation energy Ei is 0.15 eV between room temperature and 95 °C. The activation energy of boron decreases from 0.37 eV as the concentration of boron increases [19 atoms cm−3, the activation energy of boron was reduced to 0.15 eV, which is consistent with the Pearson–Bardeen curve applied in diamond by Lagrange et al. [Here,  ionized . In the perature . This inncreases . When the et al. . It was h(x) is expressed as:V(x) is the channel potential, biV is the built-in voltage, GV is the gate voltage, and In addition to the increase in channel conductance, there was also an increase in gate leakage current with increasing temperature at low drain-source voltage. The gate leakage current is thought to be due to tunneling that occurs when the insulating film becomes thin. In the case of the BDD SGFETs, the thickness of depletion layer I-V characteristics of C-O BDD SGFETs at room temperature and 80 °C when using Ag/AgCl electrode as a gate electrode, and I-V characteristics and pH sensitivity of the C-O BDD SGFET with Ag/AgCl at room temperature after pH sensitivity measurement between room temperature and 80 °C. The C-O BDD SGFET was highly pH-sensitive in the acidic region and medium pH-sensitive in the alkaline region, as with the initial room temperature measurement.I-V characteristics combined with a highly pH-sensitive vessel gate for application in the food industry at room temperature and 80 °C. The C-O BDD SGFETs were able to operate in a harsh environment of 95 °C. The transconductance increased and the gate leakage current increased as the temperature of the solution increased because of the activated boron. The C-O BDD SGFETs were highly pH-sensitive in the acidic region and medium pH-sensitive in the alkaline region at room temperature, however, they became pH insensitive to 4.27 mV/pH at high temperatures. By combining the pH-sensitive vessel gate and the pH-insensitive C-O BDD SGFETs, a system with a high pH sensitivity of −54.6 mV/pH was realized at 80 °C.


The characters of the micelles were studied by dynamic light scattering (DLS), TEM, pH titration, and drug release detection. It was found that the hydrophilic micelles could deliver bedaquiline, a hydrophobic antibacterial agent on S. mutans, in acidic environments and in mature biofilm. No cytotoxic effect on the periodontal cells was detected within 48 h. This pH-responsive micelle, being able to load hydrophobic antibacterial agent, has good clinical application potential in preventing dental caries.The bactericidal effect on biofilm is the main challenge currently faced by antibacterial agents. Nanoscale drug-delivery materials can enhance biofilm penetrability and drug bioavailability, and have significant applications in the biomedical field. Dental caries is a typical biofilm-related disease, and the acidification of biofilm pH is closely related to the development of dental caries. In this study, a pH-responsive core-shell nano micelle (mPEG-b-PDPA) capable of loading hydrophobic antibacterial agents was synthesized and characterized, including its ability to deliver antibacterial agents within an acidic biofilm. The molecular structure of this diblock copolymer was determined by hydrogen-1 nuclear magnetic resonance ( The oral cavity harbors an extremely sophisticated microbial environment in nature. As many as 700 strains live on hard and soft tissue surfaces. Maturation of biofilm and the imbalance of microecology contribute to the development of oral diseases . Dental Staphylococcal biofilms. Another group of researchers  suspension and biofilm generation in acidic environment (pH 5) but has no effect in neutral environment (Bedaquiline is an inhibitor that targets an acid-resistant protein H+-ATPase . Resultsironment . Besides1H-NMR), gel permeation chromatography (GPC), critical micelles concentrations (CMC), dynamic light scattering (DLS), and transmission electron microscopy (TEM). The cytotoxic effect on the periodontal cells and the antibacterial effect of micelles on S. mutans and the bactericidal effect on mature S. mutans biofilm were determined.Therefore, in the present study, a pH-responsive nano micelle [methoxypolyethylene glycol-b-poly-2-(diisopropylamino) ethyl methacrylate (mPEG-b-PDPA)] with and without bedaquiline loading was synthesized using ATRP . Its physical characterization was carried out by Hydrogen-1 nuclear magnetic resonance (–1); 2,2-dipyridyl ; cuprous bromide ; N,N,N′,N′,N′-pentamethyldiethylenetriamine ; dichloromethane (DCM); triethylamine; tetrahydrofuran (THF); and phosphotungstic acid were purchased from Macklin . 2-(Diisopropylamino) ethyl methacrylate (DPA) was purchased from Sigma-Aldrich . Bedaquiline was purchased from MedChemExpress (United States).Methoxypolyethylene glycol-OH ; α-bromoisobutyryl bromide  dropwise, while stirring it for 12 h to volatilize the THF. The micelles were further purified by dialyzing against Milli-Q water (MWCO 3500 Da) and the precipitate was collected by centrifugation . The storage micelle dispersion solution was prepared by adding 400 μl Milli-Q water (free micelles: 25 mg ml–1 mPEG-b-PDPA). The bedaquiline-loading micelles (10 mg ml–1 bedaquiline if it was completely encapsulation) were prepared as follows. In brief, 4 mg of bedaquiline was dissolved in 1 ml THF solution containing 10 mg of mPEG-b-PDPA diblock copolymer, followed by adding this solution dropwise into 4 ml PBS. The following protocol was exactly the same as those of mPEG-b-PDPA micelles’ synthesis.Typically, 10 mg of mPEG-b-PDPA diblock copolymer was dissolved in 1 ml of THF. The mPEG-b-PDPA micelles were formed by adding the solution into 4 ml PBS  of mPEG-b-PDPA polymer were acquired from GPC, THF was selected as the eluent. The hydrodynamic size diameter and zeta-potential of free micelles and bedaquiline-loading micelles at different pH values (5–7.4) were measured by dynamic light scattering .The –1) HCl to prepare a stock solution at a concentration of 5 mg ml–1. pH titration was carried out by adding 20 μl increments of 0.1 N (0.1 mol L–1) NaOH solution under stirring. The increase in pH in the range of 3–8 was monitored as a function of the total added volume of NaOH (VNaOH). The pH values were measured using pH meter.The mPEG-b-PDPA copolymer was first dissolved in 0.1 N (0.1 mol LThe CMC of the free micelles was determined by surface tension (platinum Wilhelmy plate). A set of solutions (1 ml) with different copolymer concentrations were dropped into 20 ml PBS under stirring to prepare the micelles solutions. The surface tension of micelles solutions was measured using Force Tensiometer  and the CMC were calculated using the software OneAttension.nm) of these two solutions was determined by UV-Vis spectra. Based on the standard curve of UV-Vis absorption of a set of bedaquiline THF solutions with defined concentrations, the bedaquiline-loading weight (m3) was determined. The bedaquiline-loading ratio and encapsulation efficiency were calculated as follows:Ten microliters of the bedaquiline-loading micelles and free micelles were dissolved in 1 ml of THF solution. The absorption , and then dropped on the copper grid and dried. The images of micelles were captured under TEM.The storage micelles diluted 10nm) until the cumulative release reached close to 100%.The 0.1 M PBS buffers of pH 5, 6, and 7.4 were prepared. Ten microliters of the bedaquiline-loading micelles and free micelles were separately mixed in 1 ml of PBS buffers with different pH. The release of bedaquiline was determined by UV-Vis spectra  under anaerobic condition at 37°C for 24 h. For the BHI medium for biofilm formation, 1% (w/v%) of sucrose solution was added at a ratio of 1:100. Then the absorption value at 600nm was measured to assess bacterial growth.Both the bacterial killing and the antibiofilm formation efficacy of micelles and bedaquiline-loaded micelles in pH 5 and 7 condition were assessed. The fourfold dilution of the storage micelle solution was prepared as working solution  (mPEG-b-PDPA) diblock copolymers were obtained Ka = 6.3)  .ca. 9.25 mg bedaquiline. The bedaquiline-loading ratio and encapsulation efficiency of mPEG-b-PDPA were calculated as 37 and 92.5%, respectively . This micelle displays a good hydrophobic agent encapsulation capacity (The UV-Vis spectra results showed that the bedaquiline-loading weight (m3) was 3.7 mg. It was calculated that 1 ml of storage micelles contained capacity .p < 0.05). At pH 6, the diameter of micelles with and without bedaquiline-loading increased significantly , and the diameter could not be measured at pH 5.mPEG-b-PDPA micelles were prepared with and without bedaquiline-loading. First, we measured the diameter of micelles in different pH aqueous solution using DLS . All micKa (pH < 6), the protonation of PDPA segment shifts the hydrophobic in the core to hydrophilic, the drug is released, and the spherical nano structure of micelle swells and even disassembles . Among them, the 1% bedaquiline-loaded micelles group (contain ca. 25 μg ml–1 bedaquiline) reduced the growth rate of the bacteria to half that of the control (p < 0.001). Our pervious study had found that at pH 7, bedaquiline cannot inhibit the biofilm formation of S. mutans . Therefore, we speculate that during biofilm development, when the pH of biofilm internal environment dropped to about 5, the nano micelles that had penetrated into the biofilm released the encapsulated bedaquiline and exerted a bactericidal effect. However, the pH of the suspended bacterial medium did not reach the level at which the encapsulated bedaquiline would be released, so no inhibitory effect was detected in the suspension groups. The inhibitory effect of bedaquiline-loaded micelles on S. mutans biofilm formation at neutral environment also indicates that the study nano micelles can penetrate into the biofilm and release antibacterial agent in an acidic environment.The antibacterial effect of the study micelles with and without bedaquiline loading on nhibited . In the  at pH 7 , while t. mutans . In ordeS. mutans was incubated in an acidic environment, the acidic environment itself had an inhibitory effect on the bacterial growth (see the control group in S. mutans suspension at pH 5 (ca. 2.5 μg ml–1 bedaquiline (IC50)] the growth of S. mutans in suspension was significantly reduced to half (p = 0.015). The growth of S. mutans in the 0.5% and the 1% bedaquiline-loaded micelles groups was even lower (p = 0.003 and p < 0.001). In the S. mutans biofilm model at pH 5 (p > 0.05) while the 0.5% and the 1% bedaquiline-loaded micelles groups significantly inhibited the development of biofilm (p < 0.001).When  at pH 5 , the fre at pH 5 , compareThe live/dead bacteria staining imaging of mature biofilms treated with micelles are shown in S. mutans . Third, this micelle-loading agent is a one-time release system, it is difficult to re-encapsulate agents and form micelles when the pH returns to neutral. Hence, a reversible micellar system capable of loading antibacterial agent needs further exploration.The study pH-responsive nanocarrier micelle, mPEG-b-PDPA, is simple to synthesize and can encapsulate hydrophobic antibacterial agents. Its good performance on antibacterial and bactericidal effects against S. mutans in neutral and acidic environments, and have a good bactericidal effect on mature biofilm. Furthermore, it does not have any cytotoxic effect on periodontal cells. The study findings show that it has a good potential for clinical application in dental caries prevention.To sum up, in this study a two-block micellar nanocarrier, mPEG-b-PDPA, was synthesized using a simple ATRP method. The micelle surface is positively charge and can be loaded with hydrophobic drugs. The bedaquiline-loaded nano-micelles inhibit the growth of The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.MZ, ZY, and EL conceived this study and analyzed the data. MZ conducted the experiments and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Low priority of disease prevention and health promotion in medical education may contribute to lack of lifestyle-counseling in clinical practice. Pharmacology-related knowledge is valued 5 times higher compared to lifestyle-related knowledge in examinations on noncommunicable diseases (NCDs) in undergraduate medical education in Sweden. This study aims to establish (i) whether medical licensing examinations are biased to favor pharmacology- over lifestyle-related knowledge and (ii) whether such a bias is present in both Sweden and the US.We identified 204 NCD-related questions from previous Swedish licensing examinations, and 77 cases from a U.S. question bank commonly used to prepare for the United States Medical Licensing Examination® (USMLE®) Step 3. With the help of expected correct answers, we determined distribution of points attainable for knowledge in the respective category (lifestyle / pharmacology / other) for 5 major NCDs: coronary heart disease (CHD), chronic obstructive pulmonary disease (COPD), diabetes, hypertension, and stroke.The percentage of points attainable for lifestyle-related knowledge was 6.7 (95% CI 4.1–9.3) in Sweden and 4.6 (95%CI 0.0–9.1) in the U.S. The respective percentages for pharmacology-related knowledge were 32.6 (95% CI 26.3–38.8) and 44.5 (95% CI 33.2–55.8) percent. The pharmacology vs. lifestyle-quotas were 4.9 in Sweden and 9.8 in the U.S. Likelihoods of equal emphasis on lifestyle and pharmacology in NCDs was < 0.001 in both countries.There is a marked preference for pharmacology over lifestyle in medical licensing examinations in both Sweden and the U.S. Newly qualified doctors may be inadequately prepared to address preventable causes of NCDs. There iessation . Eventuaessation , insuffiessation  and unheessation . Howeveressation . Large dessation , and betessation .One possible reason for difficulties to implement lifestyle counseling in health care might be a low priority of lifestyle issues in medical education. Earlier results indicate that pharmacology-related knowledge renders five times as many points compared to lifestyle-related knowledge in examinations on NCDs in undergraduate medical education in Sweden .In Sweden, medical education consists of 5½ years, followed by a compulsory 2-year internship. Thereafter, an exam must be passed before the medical license is provided which in turn is prerequisite for residency training. In the U.S., 4 years of medical education are followed by residency training, after the first year of which the United States Medical Licensing Examination® (USMLE®) step 3 must be passed for licensing. Part of the difference in total duration of medical training is explained by a compulsory pre-medical education at college level in the U.S.The aim of the present study is to establish (i) whether medical licensing examinations are biased to favor pharmacology- over lifestyle-related knowledge and (ii) whether such a bias is present in both Sweden and the US. Hence, the null-hypotheses to be tested are: examinations after postgraduate clinical training put an equal emphasis on lifestyle-factors and pharmacology in the context of NCDs in both Sweden and the United States.2From the Swedish national database of previous licensing examinations  we retri2.1Two authors  read all examinations and identified questions concerning lifestyle-related disease. We discriminated between three categories of knowledge: Lifestyle-related, pharmacology-related, and other. The latter category includes knowledge about pathophysiology, clinical examination, investigation, interventions other than lifestyle- or pharmacological, differential diagnoses, ethical considerations, etc. All questions concerning pharmacological treatment of the five NCDs were categorized as pharmacology related. Questions regarding lifestyle habits or health behavior change in the context of one of the five above-stated NCDs were categorized as lifestyle-related. Discrimination between respective categories was solely based on clinical experience and pedagogical expertise of raters. A third author (LJ) independently categorized all lifestyle-related questions with respect to obtainable points in the respective categories for right answers as well as individual lifestyle components in the background information. Conflicts of opinion regarding category and/or lifestyle components were resolved by discussion to agreement. A summary of principles for assessment is given in the A flow-chart of the assessment process is given in With the help of expected correct answers, we determined distribution of points attainable for knowledge in the respective category (lifestyle / pharmacology / other). For the few hybrids, i e questions that contained elements of both lifestyle and pharmacology, proportions of available points within the respective category were counted e.g., a 2-point multiple essay questions with one expected answer in each category rendered 1 point for both lifestyle and pharmacology. A multiple-choice question with 1 correct answer and 2 distractors within pharmacology and 2 further distractors in lifestyle would have rendered 0,6 points for pharmacology and 0,4 points for lifestyle .To further study the relative importance given to individual components of lifestyle we also categorized information given in vignettes regarding medication compliance, use of illicit drugs, alcohol, smoking, food habits, sleep habits, physical activity, stress management and - as a surrogate measure of energy balance - the presence or absence of obesity. As “genes vs. lifestyle” is a common topic in NCDs we also registered whether information regarding heredity was provided.2.2Most of the studied Swedish licensing examinations consisted of multiple essay questions with incremental provision of background information in the vignette for each subsequent question. For the purpose of testing the hypothesis of equal weight given to pharmacology-and lifestyle-related knowledge  for all statistical evaluations.To determine the likelihood of equal weight for lifestyle- and pharmacology-related knowledge, we calculated the difference between obtainable points in respective category (lifestyle vs pharmacology) for questions regarding NCDs in Sweden and the U.S. We used the sign test in the PROC UNIVARIATE procedure to obtain respective P-values for the hypothesis of equal or higher number of obtainable points for LM-knowledge compared to pharmacology-related knowledge.To assess the relative importance of lifestyle and pharmacology we noted the percentage of obtainable points for all questions regarding the 5 NCDs in both Sweden and the U.S. Comparison of means aggregated over all 5 NCDs was performed by T-test with PROC MEANS procedure.2.4This study did not involve human participants and is therefore exempt from formal ethics review.3Except for COPD, questions concerning NCDs favored pharmacology-related knowledge. The percentage of points attainable for lifestyle-related knowledge was 6.7 (95% CI 4.1–9.3) in Sweden and 4.6 (95%CI 0.0–9.1) in the U.S. The respective percentages for pharmacology-related knowledge were 32.6 (95% CI 26.3–38.8) and 44.5 (95% CI 33.2–55.8) percent. The pharmacology vs. lifestyle-quotas were 4.9 in Sweden and 9.8 in the U.S.Likelihoods of equal emphasis on lifestyle and pharmacology in NCDs is given in Distribution of points awarded in the respective categories is given in An overview of information on individual lifestyle components provided in vignettes is given in Stress-management, sleep and food habits are mentioned in < 10% of the vignettes in both countries, use of illicit drugs and medication compliance receive low attention in Sweden. Smoking is the only lifestyle component that is mentioned in more than half of the vignettes in both countries.4The null-hypothesis of an equal emphasis on lifestyle- and pharmacology-related knowledge in licensing examinations could not be confirmed, as there were substantial differences in both countries when questions about all five NCDs were considered together. The multiple at which lifestyle-related knowledge was discounted in postgraduate examinations in Sweden is almost identical with that which was previously found in undergraduate examinations on national level  . Contentforms of assessment influence students’ learning and/or performance has been studied extensively  may raise questions about the reliability of the results. BK and LJ are experienced clinicians in internal medicine and general practice, respectively. The 4 co-authors have a combined experience of several decades of teaching in higher medical education. Principles for assessment are provided in 5Our results indicate that both Swedish and U.S. medical licensing examinations put a marked emphasis on pharmacology in the management of lifestyle related NCDs. This may in turn mold future medical doctors to focus on pharmacological interventions and give lower priority to health promotion.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.B. Krachler: Conceptualization, Methodology, Formal analysis, Writing - original draft. L. Jerdén: Methodology, Formal analysis, Writing - review & editing. H. Tönnesen: Methodology, Writing - review & editing. C. Lindén: Conceptualization, Methodology, Formal analysis, Writing - review & editing.


The primary endpoint was 90-day cardiovascular follow-up rates within our health system. In the overall cohort, the mean age of patients was 69.3 years [SD 14.7] and 60.7%  were male. In the pre-order era, 90-day follow-up was 56.7 ± 0.4%  and increased to 67.9 ± 0.3%  in the EMR-order era. The use of the EMR follow-up order was independently associated with increased outpatient follow-up within 90 days after adjusting for patient demographics and payor status . The 30-day readmission rate in the pre-order era was 12.8%  compared with 13.7%  in the EMR-order era. An EMR-based appointment order for follow-up appointment scheduling was associated with increased cardiovascular medicine follow-up, but was not associated with an observed reduction in 30-day readmission rates.Outpatient follow-up after hospital discharge improves continuity of care and reduces readmissions, but rates of follow-up remain low. It is not known whether electronic medical record (EMR)-based tools improve follow-up. The aim of this study was to determine if an EMR-based order to secure cardiology follow-up appointments at hospital discharge would improve follow-up rates and hospital readmission rates. A pre-post interventional study was conducted and evaluated 39,209 cardiovascular medicine discharges within an academic center between 2012 and 2017. Follow-up rates and readmission rates were compared during 2 years prior to EMR-order implementation (pre-order era 2012–2013,  One-fifth of Medicare patients are readmitted within 30 days of the index hospitalization2. Additionally, the median 30-day mortality rate after hospital discharge among Medicare patients is 10–15% for stroke, acute myocardial infarction (AMI), heart failure, and pneumonia3.The period after hospitalization has been well documented as a high-risk period during which patients are vulnerable to adverse health outcomes including readmission and death4. The follow-up appointment offers an opportunity to provide additional counseling, coordinate recommendations from providers, and ensure proper medication regimen and compliance5. Multiple studies have demonstrated that patients who have an early follow-up after discharge have a lower risk of 30-day readmission6. Additionally, a recent study showed that 30% of AMI patients who were lost to follow-up had higher mortality rates7.During this period, timely follow-up after hospital discharge improves transitions in care, reduces readmissions, and may improve mortality8. One key factor that has been associated with a higher rate of follow-up is scheduling appointments prior to discharge9. However, a Yale-New Haven hospital study suggests that only one-third of patients have scheduled follow-up appointments at the time of discharge10. In addition, meaningful interventions have been largely unsuccessful to improve follow-up rates11.Though multiple observational studies have shown the benefit of follow-up after hospitalization, rates of follow-up remain low. Only 50% of Medicare patients had a follow-up visit with a physician before readmission12. EMR systems can play a pivotal role in designing comprehensive systems of care to protect our most vulnerable patients. To date, it is unknown whether EMR-based interventions can be leveraged to increase rates of follow-up after discharge. The purpose of this implementation study was to design an EMR-based appointment order to secure cardiovascular medicine follow-up appointments at the time of hospital discharge and to measure the impact on outpatient follow-up rates and 30-day readmission rates.The Health Information Technology for Economic and Clinical Health Act promoted the rapid adoption of electronic medical records (EMR). As a result, nearly all non-Federal hospitals had certified EMR technologies as of 2015 compared with ~10% in 2008n = 29,685) of the patients were white, 39.2% were female , and 62.8%  utilized Medicare for health insurance. The average age of patients was 69.3 years [SD 14.7] (Table n = 9844) of patients were white, 39.3% (n = 5055) were female and 60.8% utilized Medicare for health insurance. In the EMR-order era, the patient profile was similar. There was no difference in the population by sex  between the two eras. There was a statistically significant difference between race (P = 0.007), payor status (P < 0.001), and age (P < 0.001) in the two groups (pre-order era and EMR-order era).The study population included 39,209 patient discharges of which 12,852 discharges were in the pre-order era (2012–2013) and 26,357 discharges were in the EMR-order era (2014–2017). In the overall cohort, 75.7% (P < 0.001) demonstrating a significant increase in follow-up visits after the implementation of the EMR-based appointment order. The average rate of 90-day follow-up was stable at 56.7 ± 0.4% in the pre-order era and then steadily increased from 66.2 ± 0.59% in 2014 to 66.8 ± 0.6% in 2015 to 68.3 ± 0.6% in 2016 to 70.1 ± 0.6% in 2017 (P trend < 0.001) during the EMR-order era. During the EMR-order era, utilization of the order by providers increased from 49.9 ± 0.6% of discharges in 2014 to 72.1 ± 0.6% in 2015 to 75.0 ± 0.5% in 2016 to 76.7 ± 0.5% of discharges in 2017 (P trend < 0.001) , the 90-day follow-up rate was 56.7 ± 0.4% . In the EMR-order era (2014–2017), the average rate of 90-day follow-up was 67.9 ± 0.3% , commercial insurance , other insurance , age  and use of the EMR-based appointment order .  in the rate of 90-day follow-up after the implementation of the EMR-based appointment order. After adjustment for seasonality through a Fourier term (with no issue on over-dispersion and autocorrelation detected), the impact of the EMR-based appointment order on 90-day follow-up rates was unchanged , with an estimated increase of 4.0% in the count of 90-day follow-up  for a 100 count increase in EMR-order use.The unadjusted segmented Poisson regression model for 90-day follow-up over 6 years showed a 14% increase (relative risk (RR) 1.14, 95% CI 1.09–1.19, P < 0.001) in the EMR-order era. The 30-day readmission rate in the pre-order era was 12.8 ± 0.29%  compared with 13.7 ± 0.21%  in the EMR-order era. In patients who had a 30-day follow-up visit, the post-follow-up readmission rate up to 30 days post discharge was 6.4% (496 of 7793) in the pre-order era and 7.5%  in the EMR-order era indicating a direct relationship between overall follow-up and overall readmission rates.The 30-day follow-up rate in the pre-order era was 36.2 ± 0.4%  and increased to 46.8 ± 0.3% , indicating an increase in the rate of 30-day readmissions after the implementation of the EMR-based appointment order. After adjustment for seasonality through a Fourier term, the impact of the order on 30-day readmission rates was unchanged . The direct effect of order utilization on readmissions (without mediation) was estimated to be 0.05 , P < 0.001), demonstrating an inconsistent mediation of follow-up on readmission. The total effect (indirect + direct) of order utilization on monthly readmission was 0.01 , P < 0.001). Based on the indirect effect, among patients who followed up within 30 days, the probability of readmission was 3.6% lower than the probability of no readmission when the EMR-based appointment order was utilized.The bootstrapped mediation analysis showed that the indirect effect of order utilization on readmissions (considering follow-up prior to readmission as a mediator) was estimated to be −0.04 , N = 10,500), the overall follow-up rate within 1–7 days was 44.0 ± 0.9% . The 7-day follow-up rate among APP in the pre-order era was 42.6 ± 2.2% (825 of 1937) and increased to 44.4 ± 1.1%  in the EMR-order era. In addition, the overall 30-day readmission rate was 14.5 ± 0.7 %  among APP visits. Specifically, it was 14.8 ± 1.6% (287 of 1937) in the pre-order era compared with 14.4 ± 0.7%  in the EMR-order era. Regression models adjusting for demographics and insurance information indicate that there was a strong association between the 7-day follow-up and the 30-day readmission in the APP group , P < 0.001; logistic regression model: OR and 95% CI, 0.79 , P < 0.001).Among advanced practice provider (APP) visits  telephone calls were able to secure a follow-up appointment within 1 week of an emergency department visit13. The EMR-based appointment order streamlined the scheduling process and significantly reduced the logistical barriers to obtain a scheduled appointment ultimately leading to improved follow-up rates.Observational studies have demonstrated that follow-up after hospital discharge is important, but multiple barriers including logistic, financial, transportation, and socioeconomic inequalities exist in our fragmented health care system making this a challenging task for patients14. An analysis of 546,841 index admissions with a principal diagnosis of heart failure in the Nationwide Readmissions Database from 2013 to 2014 showed that patients in the lowest income quartile had a higher readmission rate when compared with those in the highest income quartiles. These findings were confirmed with a multivariable analysis 16. This study found that a simple EMR-based appointment order improved follow-up rates in all socioeconomic and racial groups. In fact, utilization of the order was the strongest predictor of follow-up when compared with key demographic and socioeconomic factors. Further analysis is needed to determine the order utilization and improvement in follow-up rates stratified by key demographic subgroups. EMR-based interventions can serve as powerful tools to provide benefit to all patients irrespective of socioeconomic status.Patients in a lower socioeconomic status are more likely to be readmitted after the index hospitalization17.The EMR-based order described in this study was implemented in ~25,000 patients over a 4-year period with increasing utilization by providers. The rapid and sustained provider adoption suggests that EMR-based scheduling was favored by providers as it was more efficient, user-friendly, and successful at scheduling follow-up appointments when compared with the traditional model where the primary team has to call and request the appointment. This simple and highly effective intervention can be reproduced and implemented in individual health systems. Due to the promising results of the intervention within the cardiovascular medicine service lines, the EMR-based order has been adopted across all specialties in the entire Cleveland Clinic Health System. Implementation of EMR-based scheduling orders should be explored in all specialties and hospital settings irrespective of the specific EMR that is utilized8. Because of the observational benefits of follow-up after hospitalization on readmissions, many proposed interventions to improve follow-up have been implemented18. During this same period of time, the adoption of EMR systems among US hospitals has been associated with a slight increase in readmissions19. This study showed that a simple EMR-based appointment order successfully increased follow-up rates, but also led to an increase in readmission rates. The reason for increased readmissions is likely multifactorial and includes earlier recognition of illness at the time of the office visit, the need to manage acute exacerbations , and the generally increased connectivity to the health system resulting in more utilization of care. Furthermore, the follow-up visit was shown in the mediation analysis to be an important contributor to readmissions. Often, sound and meaningful interventions that can improve quality of care may not translate to reductions in readmissions rates as this metric does not fully account for a paradoxical yet competing risk of death20. This study highlights the need to reexamine the intense focus on readmissions as a primary metric21.Since Jencks et al. published their landmark study showing that 19.6% of Medicare beneficiaries were readmitted within 30 days, there has been an intense effort to reduce this costly burden including the creation of Hospital Readmissions Reduction Program under the Center for Medicare and Medicaid Services22. The definition of follow-up in this study was limited to visits with cardiovascular medicine physicians, nurse practitioners, or physician assistants within one health system. Therefore, visits with a non-cardiovascular medicine health professional were not considered in the observed follow-up rates. Additionally, follow-up with providers outside of the health system was not included as data was unavailable. It is possible that if these additional visits were included, the follow-up rates would be higher. The fraction of Medicaid patients was notably different between pre-order (5.9%) and EMR-order (9.7%) eras. The increase in Medicaid patients was likely reflective of the Ohio Medicaid Expansion during the course of the study period. Since 2014, over 1.26 million people enrolled in Ohio Medicaid through the Affordable Care Act-driven expansion23. Traditionally, lower rates of follow-up have been observed in Medicaid populations, but this study found that the implementation of the EMR-based appointment order was associated with improved Medicaid follow-up rates in the EMR-order era14. The study focused on common demographic variables. Clinical data including primary diagnosis and co-morbidities were not included in order to enhance the generalizability and scalability of this model. All-cause readmissions were included in this study and it is possible some of these admissions were planned readmissions. Impact on mortality was not possible with the available data which could not accurately account for mortality outside of the health system. Lastly, although we strongly believe that the follow-up order was crucial to the findings detailed above, we acknowledge that our implementation study cannot definitively demonstrate a causal relationship.This study has several limitations. First, this was a single-center experience with a focus only on the cardiovascular medicine services at the Heart, Vascular, and Thoracic Institute. The pre-order era was a historical control arm. Though this pre-post study has the strength of temporality to suggest the improvement in follow-up rates was impacted by the implementation of the EMR-based appointment order, it does not control for other variables that are also changing at the same time as the implementation of the EMR orderThis study demonstrates that EMR-based interventions are associated with increased follow-up rates after hospital discharge. The available EMR infrastructure in nearly all US hospitals is a highly favorable environment for the widespread adoption of this simple strategy. An EMR-based strategy offers the promise and potential to secure follow-up rates after hospital discharge in an effective and efficient manner.7. All 39,209 discharges from the cardiovascular medicine services within the Heart, Vascular, and Thoracic Institute between 2012 and 2017 were included. No patients were excluded.The study was conducted at the Heart, Vascular, and Thoracic Institute in the Cleveland Clinic Main Campus. For each patient in the study, the following data were extracted from our EMR system and financial databases: date of index admission, date of index discharge, specific cardiovascular medicine service line, the utilization of the follow-up order, date of follow-up office visit, payor status, demographic information  and date of readmission. Race was subdivided into three major categories: white, black, other race. Age was divided into two groups: age ≥65 and age <65. Payor status was categorized as follows: Medicare, Medicaid, commercial insurance, other insurance . All readmissions, planned and unplanned, for any reason to any hospital within the health system  within 30 days of the index hospitalization were identified using the institutional billing system. Readmissions to hospitals outside of our health system were not available and not included in the analysis. An internal audit of the institutional readmission tracking system has shown that 78.3% of all readmissions are captured by this methodBetween January 1, 2012, and December 31, 2013, the scheduling of follow-up appointments was at the discretion of the primary clinical team. The EMR-based appointment order was created to collect information about the patient’s follow-up needs and was implemented on January 1, 2014. The providers were informed and educated about the new process through emails and announcements at staff meetings. There was no soft roll-out period or best practice advisory fire guiding that was implemented. The order collected the following information: reason for follow-up, request for a specific cardiovascular medicine provider (within 14–30 days), request for whether follow-up with a nurse practitioner or physician assistant was needed between 4 and 7 days. All providers were able to search for the order within Epic, the EMR system used at Cleveland Clinic. Once the order was entered by a provider, it was routed to an Epic pool the HVTI Scheduling Office uses to schedule the follow-up appointment. The follow-up appointment is scheduled in Epic’s standard patient scheduling module called Epic Cadence. The scheduling office was available to execute this process from Monday through Saturday. After the appointment was made, a text page was sent to the ordering provider and the information was automatically entered into the patient discharge instructions , the trend of 90-day follow-up rates over time (2012–2017), the risk-adjusted association of EMR follow-up with 90-day follow-up rates, and 30-day readmission rates presented in a pre-post format and interrupted time series. The relationship between EMR-order utilization, 30-day follow-up rates, and 30-day readmission rates was studied to understand causality using a bootstrapped mediation analysis.24. The interrupted time series regression was then extended to use the monthly counts of EMR-order use, for assessing the effect of EMR-order use on 90-day follow-up.Demographic information is reported as either mean (one SD) or frequency (percentage). Categorical variables were analyzed with a Pearson chi-square test. The unadjusted rate of 90-day follow-up in the pre-order era vs EMR-order era was compared via a Pearson chi-square test. A Kendall Tau B test of trend was used to assess trends in study order utilization over time and trends in 90-day follow-up rates over time in the EMR-order era. Multivariable logistic regression modeling was used to examine the risk-adjusted association of the EMR follow-up order with 90-day follow-up during the EMR-order era. To match the pre-post interventional study design, a segmented time series was created using the monthly data of 90-day follow-up across the pre-order and EMR-order eras, and a segmented Poisson regression model was fit using the original counts and the offset variable of the age-standardized patient population on the monthly scaleThe association between 30-day follow-up rates and 30-day readmission rates was assessed with a Pearson chi-square test. Two analyses were conducted examining the effect of the EMR order on 30-day readmissions: interrupted time series regression model on the monthly 30-day readmission data and a bootstrapped mediation analysis with multivariable logistic regression adjusting for demographics, whereby follow-up status was the mediator. To achieve a casual sequence, when a patient had a follow-up appointment visit that occurred after the day of readmission, the follow-up status was redefined as “no follow-up”.P < 0.05 indicates statistical significance for all comparisons. This study was approved by the institutional review board at the Cleveland Clinic and a waiver of written informed consent was provided.All analyses were performed with Statistical Package for the Social Sciences (SPSS) software platform  and the open-source R software. Further information on research design is available in the Supplementary InformationReporting Summary


Herein, we present an easy-to-use alternative that involves the combination of a new, methylated building block  with vacuum drying. Condensation of Me3TFB with 1,4-phenylenediamine (PA) or benzidine (BD) resulted in imine-linked 2D COFs (Me3TFB-PA and Me3TFB-BD) with higher degrees of crystallinity and higher BET surface areas compared to their non-methylated counterparts (TFB-PA and TFB-BD). This was rationalized by density functional theory computations. Additionally, the methylated COFs are less prone to pore collapse when subjected to vacuum drying and their BET surface area was found to remain stable for at least four weeks. Within the context of their applicability as sensors, we also studied the influence of hydrochloric acid vapour on the optical and structural properties of all COFs. Upon acid exposure their colour and absorbance spectra changed, making them indeed suitable for acid detection. Infrared spectroscopy revealed that the colour change is likely attributed to the cleavage of imine bonds, which are only partially restored after ammonia exposure. While this limits their application as reusable sensors, our work presents a facile method to increase the robustness of commonly known COFs.Covalent Organic Frameworks (COFs) are thermally and chemically stable, nanoporous materials with high surface areas, making them interesting for a large variety of applications including energy storage, gas separation, catalysis and chemical sensing. However, pore blocking and pore collapse may limit their performance. Reducing the capillary forces by using solvents with low surface tension, like supercritical CO Methylation of aldehyde nodes in Covalent Organic Frameworks leads to enhanced BET surface areas and reduced pore collapse compared to their non-methylated counterparts, which has been rationalized by DFT computations. This enables error correction, leading to crystalline frameworks with a long-range order. The symmetry of the building blocks is determining the crystal structure. In the case of 2D COFs, the third dimension is formed by π–π stacking. The layers usually stack in an eclipsed structure leading to channels in the framework, which results in high surface areas. These high surface areas are of interest for, amongst others, energy storage, gas separation, catalysis, and chemical sensing.3,6–8 The variability in organic building blocks in terms of functional groups or symmetry allow a high control over the COF properties and thus leads to tailor-made materials. However, the boronic esters used to make the first COFs were susceptible to hydrolysis. This has been improved by using imine-linked COFs or even more stable β-ketoenamine COFs. The latter first forms imine bonds, which then tautomerise irreversibly into the β-ketoenamine linkage. The irreversible tautomerisation results in a lower crystallinity and surface area compared to their imine counterparts.2 The chemical stability of COFs does not only depend on the type of linking chemistry, but can also be affected by the nature of the core of the organic building blocks. Ma et al.9 reported two highly porous 3D COFs of terephthalaldehyde or 4,4′-biphenyldicarboxaldehyde and a tetrahedral amine with isopropyl groups in ortho position. Isopropyl groups are hydrophobic and their steric demand protects the imine groups from hydrolysis. The COFs are reported to be stable in acidic and strong alkaline solutions without a decrease in surface area and crystallinity. Such stability was also achieved by methoxy substitution of the aldehyde node.10 In another study, Wang et al.11 reported enhanced BET surface areas for ortho methoxy-substituted aldehyde nodes and meta methyl-substituted amine linkers.Covalent Organic Frameworks (COFs) are a class of nanoporous materials first discovered in 2005 by Yaghi and co-workers.12 Capillary forces increase with decreasing pore size and COFs with typical pore sizes around a few nanometres are therefore subjected to strong capillary forces. To overcome pore collapse, milder activation methods such as supercritical CO2 drying12 or washing with ultra-low surface tension solvents prior to drying13 were suggested to keep the framework intact upon activation. Feriante et al.12 and Zhu et al.13 both reported methoxy-functionalised, imine-based COFs, which were less prone to pore collapse compared to the non-methoxylated equivalent.COF stability is not only about the chemical stability of the functional groups involved, but also about the stability of the physical properties such as pore size, pore shape and surface area. It has been identified that the conditions for work-up and COF activation are crucial to get accessible pores and high surface areas. After synthesis, COFs are most commonly washed with several solvents to remove impurities and water from the pores prior to drying. Vacuum drying has often been used, but was identified to induce partial pore collapse due to capillary forces.In the current paper, we introduce methyl groups in the synthesis of imine-based COFs as alternative to the bulky isopropyl and the smaller methoxy groups. Depending on the COF application, the use of this smaller – but still hydrophobic – group can be advantageous as it has less impact on the pore size.3TFB) as building block for two novel COFs, namely Me3TFB-PA and Me3TFB-BD, which are compared with two reference COFs TFB-PA6 and TFB-BD.14,15 The methylation significantly increases the BET surface area by a factor of 1.8 and 1.4, respectively. The physical stability of the framework towards pore collapse during vacuum activation also profits from the methylation. This work shows a facile approach in stabilizing COFs by methylating the aldehyde. The novel COFs maintain their high surface area on the bench for at least four weeks. Finally, the effect of acid vapour on the optical and structural properties of the COFs was studied.Herein, we present 2,4,6-trimethylbenzene-1,3,5-tricarb-aldehyde (Me3TFB) were condensed with two different amines, namely 1,4-phenylenediamine (PA) and benzidine (BD) (3TFB-PA and Me3TFB-BD) and their non-methylated equivalents (TFB-PA and TFB-BD). Each COF has been synthesised and characterised thrice. We selected this set of COFs, because TFB-PA and TFB-BD have been widely studied in literature.6,14–16 The synthesis was carried out at 70 °C and atmospheric pressure with acetic acid as catalyst and water to enhance dynamic covalent chemistry in a mixture of mesitylene : 1,4-dioxane 1 : 4 v/v for three days.17 A washing protocol from Vitaku et al. has been used for the work-up.18 Afterwards, half of the obtained powder was dried overnight in a vacuum-oven at 120 °C and the other half at 120 °C in a regular, air-ventilated oven before the COFs were characterised. The characterisation for the reference COFs can be found in the ESI  and 2,4,6-trimethyl-benzene-1,3,5-tricarbaldehyde (Meine (BD)  to yield3TFB-PA shows a characteristic imine stretch at 1623 cm−1, demonstrating a successful reaction. A weak band at 1693 cm−1 indicates low fractions of aldehyde bonds. This IR characteristic has also been reported by others and may origin from unreacted side-groups at the outside of the 2D polymeric sheet.12,13,19FT-IR spectroscopy on the novel COFs confirmed the formation of imine bonds . Me3TFB-13C cross-polarization magic angle spinning solid-state NMR (13C CPMAS ssNMR)   as the s3TFB-BD COF with an imine band at 1627 cm−1 and a small carbonyl stretch at 1693 cm−1  was performed on all samples to determine the crystallinity of the materials. The obtained spectra were compared to the simulated diffraction patterns of optimized crystal structures obtained from density-functional theory (DFT) calculations  was calculated using the Rouquerol criteria24 to apply the BET theory to microporous materials. For all samples of the same COF, the same pressure range was used for the linear regression. Afterwards, the obtained BET surface areas were averaged. For Me3TFB-PA SBET was found to be 1877 ± 20 m2 g−1. To determine the pore size distribution, HS-2D-NLDFT analysis was carried out using the isotherm data and a model with cylindrical micropores for carbon surfaces with N2. Me3TFB-PA COF shows a narrow pore size distribution with a clear maximum at 1.9 nm . The step in the isotherm at p/p0 = 0.1 indicates capillary condensation. The lack of hysteresis is in line with the cylindrical shape of the pores.22 The pore volume at p/p0 = 0.95 and BET surface areas were measured to be 1.24 ± 0.03 cm3 g−1 and 2115 ± 50 m2 g−1, respectively, while the pore size distribution has a narrow maximum at 2.7 nm  after vacuum activation , while both activation processes are equally good for Me3TFB-BD (retained SBET 97 ± 15%). In other words, the BET surface areas increase by a factor of 2.8 and 1.4 for Me3TFB-PA and Me3TFB-BD, respectively. The methylated COFs are hence less prone to pore collapse.To study whether the addition of methyl groups also results in a more stable framework, we studied the pore collapse by re-measuring the BET surface area of all COFs after vacuum activation . To correct for the difference in BET surface area due to different pore sizes, the retained BET surface area has been calculated as the ratio of the vacuum-activated tivation . The ret2 g−1 in the first four weeks, which means only 11% of the original BET surface area are accessible after one month. Therefore, we investigated the air stability of the other three COFs as well, by leaving the oven-activated samples for four weeks in an open vial on the bench, before determining the BET surface area again. These three COFs did not show such a large drop in the SBET values. Instead, on average a slight increase is observed (up to 8% for Me3TFB-BD), although the differences are within the standard deviation. In any case, as no decrease is observed, we conclude that TFB-BD, Me3TFB-PA, and Me3TFB-BD are stable for the reported conditions. Again, this shows that the introduction of methyl groups stabilizes the phenylenediamine COF.In addition, we noticed that the BET surface area of TFB-PA decreases already drastically to 113 ± 53 met al. observed a solvatochromic effect, which has been visible in diffuse reflectance spectroscopy, before employing the pyrene-containing COF for the development of a humidity sensor.8 Several studies report on COF-based chemosensors for the detection of gaseous hydrochloric acid,25,26 and a series of aqueous Brønsted acids.26,27 Recently, Yang et al. have studied the effect of imine-protonation on the optical properties of three imine-linked COFs. It was found that all COFs showed a bathochromic shift after acid exposure due to the protonation of the imine bond.28 Based on both findings, we looked into the absorption spectra of Me3TFB-PA and Me3TFB-BD and their response towards hydrochloric acid vapour. To this end, samples of the new COFs were pressed as a thin film between two glass plates. Absorption spectra were collected before treatment with acid by measuring the samples in an integrating sphere and collecting diffuse reflectance and diffuse transmission to calculate the absorbance. Both absorbance spectra have an inverse S-curve shape, start absorbing between 400–450 nm, and have absorption edges around 378 nm for Me3TFB-PA and 385 nm for Me3TFB-BD (3TFB-PA and 419 nm for Me3TFB-BD COF).The optical properties of some COFs are reported to respond to changes in acidity and solvent, enabling them to be used as chemical sensors. In more detail, Ascherl e3TFB-BD . To facie3TFB-BD , which wAs the two-plate configuration does not allow vapour exposure experiments, a new sample was taken from the same synthetic COF batch, which was subjected to hydrochloric acid vapour for 30 minutes. Within minutes, a colour change  was observed by the naked eye , which w3TFB-PA COF, although the first derivative indicates a wider range in which the absorbance changes and the absorption edge is red shifted with respect to the acid-exposed sample. For Me3TFB-BD COF, the spectrum also shifts back to higher wavelength, but even wider. The first derivative indicates two small minima with shifted positions that do not overlap with the original minimum, so the acid has an impact on the COF structure. The absorbance spectra for the non-methylated COFs can be found in the ESI  and 1685 cm−1 (carbonyl stretching vibration) are visible, indicating a partial recovery of the imine bonds.We speculate that the acid protonates the nitrogen atom . In a se3TFB-PA and Me3TFB-BD. They have been compared with non-methylated reference COFs regarding their BET surface area, pore collapse and their applicability as optical acid sensors. With a facile synthetic approach, which does not require any specialized equipment, we found that methylation of the aldehyde node results in enhanced BET surface areas up to ca. 2000 m2 g−1 and high degrees of crystallinity compared to their non-methylated equivalents. The obtained BET surface areas are comparable to the ones achieved by supercritical CO2 activation. The higher surface areas and crystallinity are in line with DFT-calculated reaction rates. With respect to TFB, Me3TFB loses symmetry and its full conjugation due to the ortho-substituted methyl groups. This leads to a smaller rotation barrier towards the favoured C3 conformation for crystallisation and therefore to higher reaction rates. Additionally, the novel methylated COFs are less prone to pore collapse during vacuum activation and the BET surface area remains stable within at least four weeks.In conclusion, a new methyl-substituted aldehyde building block has been used for COF synthesis, resulting in two novel frameworks, namely Meortho-substituted methyl groups of the amine building block and systematically increasing the number of methyl groups on the resulting COF properties are currently under investigation.The COFs can be used as an optical sensor to detect hydrochloric acid based on a change in colour. The sensing mechanism reveals that the colour change is likely attributed to the cleavage of the imine bond, which is partially restored after exposure to ammonia vapour. Even though this limits their applicability as a reusable sensor for hydrochloric acid, our approach enables the facile synthesis of high surface area COFs that are more robust compared to commonly known COFs. The effect of L.C.P.M.d.S. and E.D. conceived the project. E.D. and M.L. designed and performed the experiments. G.L. performed the modelling. E.D., M.L. and L.C.P.M.d.S. analysed all data. L.C.P.M.d.S. guided the project. E.D. wrote the first draft, and all authors gave input.There are no conflicts to declare.NR-013-D1NR05911D-s001NR-013-D1NR05911D-s002NR-013-D1NR05911D-s003NR-013-D1NR05911D-s004NR-013-D1NR05911D-s005


Ganoderma lucidum extract is a potent traditional remedy for curing various ailments. Drying is the most important postharvest step during the processing of Ganoderma lucidum. The drying process mainly involves heat (36 h at 60 °C) and freeze-drying (36 h at −80 °C). We investigated the effects of different postharvest drying protocols on the metabolites profiling of Ganoderma lucidum using GC-MS, followed by an investigation of the anti-neuroinflammatory potential in LPS-treated BV2 microglial cells. A total of 109 primary metabolites were detected from heat and freeze-dried samples. Primary metabolite profiling showed higher levels of amino acids (17.4%) and monosaccharides (8.8%) in the heat-dried extracts, whereas high levels of organic acids (64.1%) were present in the freeze-dried samples. The enzymatic activity, such as ATP-citrate synthase, pyruvate kinase, glyceraldehyde-3-phosphatase dehydrogenase, glutamine synthase, fructose-bisphosphate aldolase, and D-3-phosphoglycerate dehydrogenase, related to the reverse tricarboxylic acid cycle were significantly high in the heat-dried samples. We also observed a decreased phosphorylation level of the MAP kinase  and NF-κB subunit p65 in the heat-dried samples of the BV2 microglia cells. The current study suggests that heat drying improves the production of ganoderic acids by the upregulation of TCA-related pathways, which, in turn, gives a significant reduction in the inflammatory response of LPS-induced BV2 cells. This may be attributed to the inhibition of NF-κB and MAP kinase signaling pathways in cells treated with heat-dried extracts. Drying is the method of removing the water content by deploying heat. It helps to preserve fresh food products by the suppression of water-linked activities. The process of food drying can be categorized into two major phenomena, i.e., in-vacuum or in-air, based on the medium amount used for dehydration. During drying, the heat can be transferred through different modes, such as radiation, convection, microwave, conduction, and solar sources. In the drying process, the transfer of heat from source to plant material and transfer of water and moisture content from plant material to the surface and surroundings take place simultaneously .In the postharvest process, drying is considered as one of the foremost energy-intensive operation. It has been applied to food products such as fruits, herbal products, and other agricultural products after harvest . ReducedAny postharvest processing method applied to foods that employ high temperatures may lead to the loss of important heat-sensitive metabolites. The use of a product-adaptive drying method is a very important step in postharvest processing. Antioxidant components such as tocopherols, ascorbic acid, carotenoids, and plant phenolics are highly sensitive to heat, and therefore, freeze-drying is generally recommended . The freFreeze-drying and conventional heat-drying methods are considered as the most efficient and effective when it comes to use in phytochemical-related industries ,11. OtheGanoderma lucidum (GL) is one of the most important species of the family Ganodermataceae and is commonly known as Lin-Zhi in China, Reishi in Japan, and Yeongji in Korea [Ganoderma lucidum extract (GL extract) could elicit innate immune responses, control cell proliferation, and cause cancer cell migration [in Korea . GL is win Korea ,15,16. Gin Korea . Previouigration ,19,20. Migration . The mosigration .Inflammation is a hallmark of many neurodegenerative diseases, and microglia regulate the immune responses in the central nervous system (CNS) . The infMAPK engages in biological responses, including cell proliferation, differentiation, and death. In MAPK signaling, three successively activated protein kinases called p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) are involved. The JNK and p38 modules activated by inflammatory cytokines or environmental stresses play an essential role in inflammation . ERK conDue to the pharmaceutical significance of GL, its fruiting bodies are cultivated on wood logs at the industrial level. During the postharvesting period, drying is considered as the most energy- and time-consuming process. The fruiting bodies are dried to enhance their shelf lives, make transportation easy, and prevent the degradation of the important bioactive compounds. In our previous study, extraction optimization and ganoderic acid content determination in heat- and freeze-dried GL fruiting bodies were performed, which showed differences in the secondary metabolite contents of the fruiting bodies dried by the two methods . SeveralThis study was performed as a continuation of our previous study on the extraction optimization and detection of ganoderic acid in both heat- and freeze-dried GL samples. In the present study, we determined the effects of two different (heat-drying and freeze-drying) postharvest drying methods used commercially in metabolite profiling and their therapeutic effects. GC-MS-based However, little is known about the metabolic constituents and the effect of the optimized ethanolic extract of heat-dried GL on neuroinflammation.p < 0.05)  . Among t < 0.05) . A totalThe heat-treated samples showed higher concentrations of ATP-citrate synthase (ACS), pyruvate kinase, glyceraldehyde-3-phosphatase dehydrogenase, glutamine synthase, fructose-bisphosphate aldolase, and D-3-phosphoglycerate dehydrogenase than the freeze-dried samples A. The toAbout 78.4% of the variance was explained by the PC1 and PC2 components based on the PCA analysis performed on a reduced dataset of 20 significant enzymes detected in the protein analysis. PC1 showed the highest values for ATPase, PK1, CAT, chorismite synthase (CS), and ACS and showed the lowest value for cystathionine beta-synthase (CBS) B. SuccinTo investigate the effect of each GL extract on the physiological activities in neuronal cells, we performed cell viability assays using BV2 cells. The cells were treated with a GL extract in a dose-dependent manner (1–100 μg/mL). We observed that low concentrations (1 μg/mL) of both the GL extract samples did not significantly affect the cytotoxicity. In some GL extracts with high concentrations (>10 μg/mL), a mild cell toxicity was recorded . This reThe MAPK signaling pathway is a key mediator in activating the production of proinflammatory cytokines secreted by the microglia ,40. To dTo determine whether the heat-dried GL extract or freeze-dried GL extract had a suppressive effect on the activation of the NF-κB signaling pathway, the level of p65 phosphorylation  was determined in the LPS-treated microglial cell line (BV2 cell line). We found that the heat-dried GL extract significantly inhibited the LPS-induced phosphorylation of p65 in this cell line in a dose-dependent manner E,F. The In this study, we determined the effects of two commonly used drying methods on the metabolites profiling and anti-neurodegenerative potential of the GL extract in LPS-treated microglia via NF-κB signaling and MAPK signaling. In NF-κB signaling, the effect of the heat-dried GL extract was concentration-dependent. As the GL extract concentration increased, the expression of phospho-p65 was inhibited. Moreover, the GL extract also had a dramatic effect on the activation of MAPKs, including p38, JNK, and ERK.GL is used as an herbal remedy for many ailments in Eastern Asia ,39,40. TWe studied the variations in GL primary metabolites by applying two different drying temperatures. Heat-drying may change the activity of certain enzymes after harvesting . On the In fungi, tricarboxylic acid (TCA) and glyoxylate cycles are important energy-generation pathways comprising two or three carbon compounds. Under limited nutrients, fungi start depending on compounds with higher molecular weights, such as fatty acids, and secondary metabolites by activating TCA and glyoxylate cycles . During Our proteomics study showed that the levels of glyoxylate cycle-related enzymes in GL were increased in response to heat , while tGanoderic acids exert anti-inflammatory activity ,53, antiNeuroinflammation is the first response to the onset and progression of neurodegenerative diseases like PD and AD. The inflammation of neurons is actively involved in the progression of disease. Neuroinflammation is a protective response in the case of injuries to neurons, and it helps to mediate and restore damaged glial and neuronal cells. However, in the chronic stage, it causes degradation of the neuronal cells. The NF-ĸB response is different at different stages of disease progression in AD; it is involved in the development of plaque and during the signaling of cytokine and inflammation during disease progression. Slowing down the activity of NF-ĸB results in decreased progression of the disease .We observed that, in NF-κB signaling, the effect of the GL extract was dependent on the concentration of the GL extract. As the concentration increased, the expression of phospho-p65 was inhibited. Moreover, the GL extract also had a inhibitory effect on the activation of MAPK, including p38, JNK, and ERK . We obsev/v/v), including ribitol and 5α-cholestane as the internal standard. Extraction was performed at 37 °C, and the samples were mixed at 1200 rpm for 30 min. The extracted solution was centrifuged for 3 min at 13,000 rpm. The polar phase was collected into a new tube, and distilled water was added. Samples were centrifuged at 13,000 rpm for 3 min, and the methanol/water phases were dried using a freeze dryer. The derivative process was performed using methoxyamine hydrochloride (methoxime) and trimethylsilyl [GL samples were procured from Korea Ginseng Corporation . Heat- and freeze-dried samples were dehydrated for 36 h at 60 °C and −80 °C, respectively. For metabolite profiling, the freeze-dried samples (10 mg) were dissolved in the extraction solvent mixture of water, methanol, and chloroform . The starting temperature of the oven was 80 °C for 2 min and was then increased by 15 °C per min to attain 320 °C for 10 min. The transfer line temperature was programmed to be 250 °C, and the ion source temperature was 200 °C. TOF-MS acquisition was delayed for 180 s, with an acquisition rate of 20 spectra per second. The detector voltage and electron energy were set at 1650 V and 70 eV, respectively [An analysis of the primary metabolites was performed using GC-TOF-MS . The sample was injected in a split mode (1:25) with an injection volume of 1 μL at 230 °C. Carrier gas (helium) flowed at a rate of 1 mL minectively .−1 and heated at 95 °C for 10 min [Approximately 40 mg of powdered GL samples were transferred to a prefilled bead tube . A 500-µL Lysis buffer , 0.4% sodium lauryl sulfate (SDS), 50-mM NaCl, and 1-mM PMSF) was added to the tube and homogenized at 30 pulses/s for 8 min, with a TissueLyser II . After heating the samples at 60 °C for 15 min, the samples were sonicated  at 30% amplitude for 2-s and 1-s on/off cycles, respectively, for 1 min. The sonicated samples were centrifuged at 13,000 rpm at 4 °C for 15 min, and the supernatants were transferred to a new tube. A bicinchoninic acid (BCA) gold protein assay kit  was used for the quantification of the crude protein. The crude protein samples (100 µg) were precipitated using 5 volumes of cold acetone and stored at −20 °C overnight. After centrifugation at 13,000 rpm, 4 °C for 15 min, the protein precipitates were collected. The pellets were resuspended in a LDS sample buffer  containing 50-mM DTT to a final concentration of 2 mg mLr 10 min .™ Mass Spectrometer  equipped with an Easy-nLC 1000 system  was used for the peptide analysis. A 5-μL peptide sample was injected and loaded onto an Acclaim PepMap™ 100 trap column . An EASY-Spray column  was used for sample separation. Solvent A (0.1% formic acid in water) and B (0.1% formic acid in ACN) at a flow rate of 300 nL min−1 were used. The gradient started with 5% solvent B for 5 min and increased to 50% by 40 min and then increased to 100% by 41 min and was maintained for another 5 min. Prior to sample loading, the trap and analytical columns were equilibrated with a 10× column volume of solvent A. The eluted peptides were ionized with a voltage of 2 kV and subjected to mass spectrometry. The peptide ions were first analyzed with a full MS scan in a range of 50–2000 m/z and a resolution of 70,000.The crude protein samples (40 µg) were separated on a NuPage Bis-Tris SDS-PAGE gel (4–12%) . Coomassie brilliant blue g-250 was used as a staining agent. The gels were subjected to a conventional in-gel digestion with minor modifications . The Q Exactive™ Hybrid Quadrupole-OrbitrapTandem mass spectra were analyzed using the SEQUEST module of Proteome Discoverer . The keyword “organism: Ganoderma”  was used to download the protein database from UniProt. Scaffold  was used to validate the MS/MS-based peptide and protein identifications. Normalized spectrum counting was performed for the semi-quantitative analysis of the proteins .4 cells/well) were seeded into a 96-well plate. BV2 cells were treated with each extract for 12 h. After adding a solubilization solution and incubating the mixture at room temperature for 16 h, the insoluble formazan was measured at 570 nm [The cytotoxicity of each extract in the BV2 cells was evaluated by the MTT assay (Promega) by following the manufacturer’s instructions. BV2 cells  for 15 min at 37 °C. BV2 cells in each well were cotreated with LPS (1 µg/mL), followed by the incubation at 37 °C for 12 h. One milliliter of supernatant was taken. The cells in each well were washed with cold 1X phosphate buffer solution, followed by the treatment with tissue lysis buffer containing 1% SDS (200 µL). Then, the cell lysate was scraped and collected in an e-tube [BV2 cells were seeded into a 6-well plate. The number of cells seeded per well of the plate was 2 × 10n e-tube .A BCA assay was used to measure the protein concentration of cell lysates. The solution was separated by SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred to a protein membrane and blocked with 5% skimmed milk in 1× phosphate-buffered saline with 2% Tween-20. The cells were then treated overnight at 4 °C with the primary antibodies (1:1000) and then with the secondary antibodies . The maximum sensitivity substrate and signal intensities were measured .The software package SIMCA-P 15.0.2  was used for the multivariate data analysis. A principal component analysis (PCA) was performed with auto-scaled data to understand an overview of the group clustering and to identify the possible outliers. Data were presented as the mean of the standard deviation. All the experiments were performed in triplicate.Among the two commonly used drying techniques we investigated, the heat-drying method was found to be the most effective method in the postharvest processing of Ganoderma lucidum. As compared to the freeze-drying method, the therapeutic potential was affected to a greater degree. This suggests that the postharvest drying process has a greater impact on the pharmacological properties of Ganoderma lucidum.The results showed that heat-drying helps to improve the metabolites content by the upregulation of the enzymes related to the TCA cycle. The heat-dried extract also performed very well against LPS-induced neuroinflammation in the BV2 cells. This suggests that the heat-dried GL extract can be used as an important drug component in slowing the progression of neurodegenerative diseases.This provides strong evidence that the postharvest drying methods can be a decisive factor in the selection of raw materials for the pharmaceutical industry. The heat-dried GL extract may play a critical role in the development of potential drugs for the neurodegenerative diseases resulting from LPS-induced neuroinflammation.


Diabetic peripheral neuropathy (DPN) is one of the most common chronic complications of diabetes, leading to disability and decreased quality of life. In past research and clinical studies, the lower limb function of DPN patients was often the principal subject of research, with little attention given to the upper limb and hand. Our goal was to assess and compare hand function between elderly diabetic patients with DPN and without DPN.  A total of 52 diabetic patients were registered and underwent hand function assessments and electrodiagnostic tests. Dynamometer, pinch meter, Semmes Weinstein monofilaments, and the Purdue Pegboard Test (PPT) were used to assess the patients' grip strength, pinch strength, tactile sensory threshold, and hand dexterity. p = 0.019; 1.50  vs. 2.50 , p < 0.001); the elderly DPN group displayed worse thumb-middle fingertip pinch strength, thumb-ring fingertip pinch strength, and thumb-little fingertip pinch strength in the nondominant hand  vs. 4.00 , p = 0.013; 2.50  vs. 3.00 , p = 0.033; 1.00  vs. 2.50 , p < 0.001). The elderly DPN group scored lower than the non-DPN group on the PPT test of assembly .  Compared with the non-DPN group, the elderly DPN group showed worse thumb-middle fingertip pinch strength and thumb-little fingertip pinch strength in the dominant hand  vs. 4.50 , ChiCTR1900025358. Motor function limitation is the principal hand dysfunction in elderly patients with DPN, which is mainly manifested as a decline in fingertip pinch strength and a decrease in hand dexterity. This trial is registered with Clinical Trial Registry no.  China has the largest number of diabetes mellitus cases and the highest annual increases in cases in the world, with 116 million cases nationwide in 2019 . ApproxiWith the increasing prevalence of diabetes and the prolongation of the life span of diabetic patients, the prevalence of diabetes in the elderly is rising . TherefoChiCTR1900025358). A total of 52 participants who met the above criteria were included  participants were age 65 years or older with a previous diagnosis of type 2 diabetes for more than 5 years; (2) their blood glucose control was basically stable, and there was no acute complications of diabetes; and (3) informed consent was obtained from all participants. The exclusion criteria included the following: (1) history of stroke, Parkinson's disease, and other central system diseases; (2) history of peripheral nervous system diseases such as carpal tunnel syndrome; (3) hands with ulcers, gout, and disability; (4) new fractures and severe muscle atrophy in the hands; (5) habit/history of alcohol abuse; (6) participants with no education; and (7) participants with severe cognitive impairment  score ≤ 18). This study was approved by the Ethics Committee of Huashan Hospital Affiliated to Fudan University (NO.2019M-006) and the Ethics Committee of Jiaozuo People's Hospital (NO.2019001) and was registered in the China Clinical Trial Registration Center  is often used to assess the tactile sensation function of the hands and feet in patients with DPN . All parGrip strength of the participants was assessed using a hydraulic hand dynamometer  with participants seated with the shoulder adducted and neutrally rotated, the elbow flexed at 90 degrees, and the forearm in the neutral position. We used a mechanical pinch-strength meter  to measure the pinch strength between the thumb and the remaining four fingers. The measurement methods were as follows: (1) thumb-index fingertip pinch strength (Pinch-I), (2) thumb-middle fingertip pinch strength (Pinch-M), (3) thumb-ring fingertip pinch strength (Pinch-R), and (4) thumb-little fingertip pinch strength (Pinch-L). Each was measured three times, and the maximum value was recorded.Hand dexterity is an important manifestation of upper limb motor function. We also assessed fine-motor-skill performance using the previously validated Purdue Pegboard Test  following a standard testing protocol . ParticiNerve conduction studies (NCS) are the current gold standard for the diagnosis of DPN . All part-test was used to compare the means of two groups, and a chi-square test was used to compare the percentages. Data of skewed distribution were presented as the median (25th-75th percentile) and tested with the Mann–Whitney U test. A value of p < 0.05 was considered statistically significant.Statistical analysis was performed using SPSS for Windows, version 21.0 . All statistics were tested for normal distribution. The continuous data of normal distribution were expressed as mean ± standard deviation, and categorical variables were presented as numbers and percentages (%). An independent sample 2, the waist-to-hip ratio was 0.93 ± 0.07, and the diabetes duration was 15.0  years. The percentage of participants who self-reported smoking, drinking, and coronary heart disease was 28.0%, 24.0%, and 40.0%, respectively. The non-DPN group had 27 participants  with an average age of 72.1 ± 5.5 years. The BMI was 25.5 ± 3.6 kg/m2, the waist-to-hip ratio was 0.90 ± 0.07, and the diabetes duration was 12.0  years. The percentage of participants who self-reported smoking, drinking, and coronary heart disease was 11.1%, 11.1%, and 37.0%, respectively. There were no significant differences in baseline data between the two groups (p > 0.05)  with an average age of 73.0 ± 5.3 years. The body mass index (BMI) was 25.6 ± 3.0 kg/m > 0.05) .p < 0.05). The conduction velocities in the median sensory nerve and median motor nerve in the nondominant hand in the DPN group were lower than those in the non-DPN group (p < 0.05). There were significant differences in four nerve conduction velocities in the lower limbs between the two groups (p < 0.05). The results showed that the median sensory nerve action potential, the peroneal nerve compound muscle action potential, and the tibial nerve compound muscle action potential in the dominant hand/lower limb in the DPN group were lower than those in the non-DPN group (p < 0.05). The median sensory nerve action potential, the median nerve compound muscle action potential, the ulnar sensory nerve action potential, and the peroneal nerve compound muscle action potential in the nondominant hand/lower limb in the DPN group were lower than those of the non-DPN group (p < 0.05). There were no significant differences in the SWME threshold between the two groups (p > 0.05)  .p > 0.05). The results showed that the thumb-middle fingertip pinch strength and thumb-little fingertip pinch strength of the DPN group were lower than those of the non-DPN group in the dominant hand (p < 0.05). There were no significant differences in the thumb-index fingertip pinch strength and thumb-ring fingertip pinch strength between the two groups in the dominant hand (p > 0.05). The thumb-middle fingertip pinch strength, thumb-ring fingertip pinch strength, and thumb-little fingertip pinch strength of the DPN group were lower than those of the non-DPN group in the nondominant hand (p < 0.05)  .p > 0.05). The results showed that the DPN group scored lower than the non-DPN group in PPT test of assembly  (= 0.032) .The study showed that elderly patients with DPN displayed worse fingertip pinch strength, especially in thumb-middle fingertip pinch strength, thumb-ring fingertip pinch strength, and thumb-little fingertip pinch strength in both dominant and nondominant hands. Weakness in pinch strength may affect the dexterity of the patient's hands.Sensory function is an important part of hand function. In the past reports, DPN caused sensory impairments such as pain, decreased vibration sensation, and loss of tactile sensation , 22. SenGrip strength is a measure of upper limb performance and is closely related to muscle strength, which is considered an important component of physical fitness and a convenient but powerful predictor of disability, morbidity, and mortality in the elderly population –29. The Pinch strength is the most frequently used hand motor function in activities of daily living, and it is also an important part of hand function. Good pinch strength is conducive to the fine motor movements of the hands. In this study, a fingertip-type pinch strength measurement method was used, and it was found that the hand pinch strength performance of the DPN group was worse than that of the non-DPN group, especially the thumb-middle fingertip pinch strength, thumb-ring fingertip pinch strength, and thumb-little fingertip pinch strength in both hands . This diReduced hand dexterity will directly affect the hand function of the elderly and indirectly lead to the decline in activities of daily living. In this study, we tested hand dexterity using PPT and found no differences between the two groups when DPN patients performed relatively simple hole-filling tasks. When patients performed assembly activities, the DPN group was worse than the non-DPN group . This reIn our clinical work, although the lower limb dysfunction of DPN patients was prominent, function problems in the upper limbs and hands could not be ignored. In the future rehabilitation training, the sensory and motor functions of the hands of patients with DPN deserve clinical attention by therapists. In particular, focusing on the patient's hand muscle strength and dexterity may improve the patient's quality of life. There are some limitations in this study. The sample is small and a cross-sectional study with a single source of cases. Therefore, the results obtained may be biased and relatively limited. In the future, large-sample, multicenter research will need to be carried out, and a control group will be set up. In addition, sensory assessment was relatively narrow. We focused only on the tactile sensation of DPN patients and did not assess patients' temperature sensation, vibration sensation, and two-point discrimination.In the elderly, limited motor function is the main hand dysfunction in patients with DPN, which is mostly manifested as a decline of fingertip pinch strength and decrease in hand dexterity.


Pneumocystis pneumonia (PCP) is a fulminant disease with an increasing incidence. The serum beta-d-glucan (BDG) assay is used as an adjunct to the diagnosis of PCP; however, the cut-off value for this assay is not well-defined, especially in the non-HIV PCP population. Therefore, we aimed to identify the assay cut-off value for this population.Non-human immunodeficiency virus (HIV) In this retrospective observational study, we reviewed the medical records of all patients (≥ 18 years old) with clinical suspicion of PCP who underwent evaluation of respiratory tract specimens between December 2008 and June 2014 at Kameda Medical Center. We created a receiver operating characteristic curve and calculated the area under the curve to determine the cut-off value for evaluating the inspection accuracy of the BDG assay.A total of 173 patients were included in the study. Fifty patients showed positive results in specimen staining, loop-mediated isothermal amplification assay, and polymerase chain reaction test, while 123 patients showed negative results. The receiver operating characteristic analyses suggested that the BDG cut-off level was 8.5 pg/mL, with a sensitivity and specificity of 76% and 76%, respectively.The Wako-BDG cut-off value for the diagnosis of non-HIV PCP is 8.5 pg/mL, which is lower than the classical cut-off value from previous studies. Clinicians should potentially consider this lower BDG cut-off value in the diagnosis and management of patients with non-HIV PCP.Trial registration: The participants were retrospectively registered.The online version contains supplementary material available at 10.1186/s12879-021-06895-x. Pneumocystis pneumonia (PCP) is a fulminant infectious disease that occurs in immunocompromised individuals . Human iPneumocystis jirovecii with staining of specimens has been the gold standard for several decades [Pneumocystis-specific polymerase chain reaction (PCR) assays have also been developed for the detection of Pneumocystis in induced sputum or bronchoalveolar lavage fluid specimens [d-glucan (BDG) assay can be used as an adjunct to the diagnosis of PCP [Pneumocystis burden [Microscopic identification of  decades . Recentlpecimens . Howeverpecimens , the sers of PCP . This tes of PCP , and shos of PCP . Howevers burden , 11. A ps burden . The sers burden , 14. HowThus, in this study, we assessed the clinical and analytical performance of the serum BDG assay in the diagnosis of PCP in non-HIV-infected patients and aimed to identify the cut-off value for this population.Pneumocystis jirovecii in respiratory specimens . Exclusion criteria were as follows (1) positive results in the HIV antibody test, (2) did not undergo serum BDG assay.We retrospectively enrolled consecutive patients (≥ 18 years old) who underwent both serum BDG assay and the evaluation of respiratory tract specimens for the diagnosis of PCP between December 2008 and June 2014 at the 917-bed Kameda Medical Center in Japan. The inclusion criteria were as follows: (1) patients (≥ 18 years old) who were suspected to have PCP based on immunocompromised status, clinical symptoms such as dyspnea, cough, and fever, and radiological findings compatible with PCP , and (2) underwent the nucleic acid amplification test  assay and/or qualitative PCR) for the detection of The serum BDG assay results, which were collected within three days before the respiratory tract specimens were tested, were used for the analysis. In addition to LAMP and/or qualitative PCR, microscopic stainings (Grocott methenamine silver stain and/or Diff-Quick™) were performed when available at the discretion of the attending physician. PCP was diagnosed based on the presence of all the following criteria: (1) positive conventional staining (Grocott methenamine silver or Diff-Quick™) or nucleic acid amplification test (LAMP or PCR) of respiratory specimens; (2) chest radiography or computed tomography (CT) findings compatible with PCP, such as bilateral ground-glass opacity; and (3) compatible clinical symptoms, including dyspnea, cough, and fever.The following patient demographics and clinical variables were collected: age, sex, underlying diseases, and serum BDG value. This retrospective cohort study was reviewed and approved by the Research Ethics Committee of Kameda Medical Center (#20-147). The informed consent was waived by the Research Ethics Committee of Kameda Medical Center because retrospective data was collected from hospital records.Limulus amoebocyte lysate reagent was then added to 200 µL of the pre-treated sample. Kinetic turbidimetry results were measured after 90 min at 37 °C using an MT-6500 toxinometer with a single extension module . The BDG concentration was calculated by comparing the gelation time to that on a calibration curve provided by the manufacturer for each lot.The WAKO β-d-Glucan test , a kinetic turbidimetric assay, was used to measure BDG levels. Briefly, 100 µL of serum was added to 900 µL of the pre-treatment solution. After incubation for 10 min at 70 °C, the sample was cooled on ice. The Pneumocystis jirovecii plasmid DNA (6 × 106 copies per tube) [The LAMP assay was conducted with the primers F3, B3, FIP, BIP, FL, and BL, as previously described , 16. Theer tube)  and negaQualitative PCR was performed using previously described primers  and a prFor the descriptive statistics of patient characteristics, categorical variables are shown as numbers (percentages) and continuous variables as medians (25th–75th percentile). The differences in background characteristics between PCP and non-PCP patients were compared using the t-test for continuous variables and the chi-square test for categorical variables. Statistical significance was defined as p < 0.05.d-glucan (< 5 pg/mL) were regarded as 0.0 pg/mL. We compared the sensitivity, specificity, positive predictive value, and negative predictive value (NPV) based on the cut-off point of this study and a previous study including patients with HIV. We obtained 95% confidence intervals of sensitivity, specificity, positive predictive value, NPV, area under the curve, and BDG cut-off point using the 2000 bootstrap procedure. Statistical analyses were performed using R software  and the pROC [To evaluate the inspection accuracy of the BDG assay, a receiver operating characteristic curve was created, and the area under the curve was calculated. The Youden Index  was thenFigure The demographic and clinical characteristics of the patients included in this study are summarized in Table The receiver operating characteristic curves constructed for BDG are shown in Fig. In this study, we evaluated the diagnostic performance of the serum Fujifilm Wako BDG assay for non-HIV-infected PCP patients. Our study findings, which were derived from 173 patients with lower airway disease who underwent evaluations for PCP, suggested a serum Wako BDG cut-off value of 8.5 pg/mL in non-HIV-infected PCP patients. In comparison with the previously reported cut-off value (31.1 pg/mL) obtained using the Fujifilm Wako-BDG assay in both HIV-PCP and non-HIV PCP populations diagnosed by microscopic staining in bronchoalveolar lavage fluid , the cutPneumocystis jirovecii. However, in recent clinical practice, clinicians have started using nucleic acid amplification test such as PCR for the detection of Pneumocystis because the sensitivity of microscopy with staining is low due to the decreased organism burden in non-HIV PCP [The results obtained in this study can be attributed to the following reasons. First, we excluded HIV-PCP patients to evaluate the serum Wako-BDG cut-off value for the diagnosis of non-HIV PCP. The serum BDG level in HIV-PCP is higher than that in non-HIV PCP because of the higher fungal burden in HIV patients, which correlates with serum BDG levels , 23. Thi-HIV PCP . Thus, oLimulus amoebocyte lysate, similar to the Fungitell assay, which is the only FDA (Food and Drug Admistration)-cleared and CE (Conformité Européenne)-marked assay worldwide. However, this assay has some advantages over the Fungitell assay, such as not being influenced by hemolytic, lipemic, or icteric samples [Our findings have important clinical implications for physicians who treat non-HIV-infected patients with PCP. The incidence and prevalence of non-HIV PCP have been increasing worldwide and considering the high in-hospital mortality rate of 30–60% associated with this form of PCP, prompt diagnosis of this condition is a critical issue that needs to be resolved . The Fuj samples ; therefoPneumocystis jirovecii. Therefore, a positive result does not necessarily indicate PCP. There have been some studies to distinguish colonization from infection using quantitative PCR, however, it is difficult to clearly distinguish between colonization and true PCP [This study had several limitations. First, the results of BDG tests could change clinicians’ decisions regarding the use of further tests to diagnose PCP. The relationship between the serum BDG level and PCP is already known ; therefotrue PCP , 28. PCRtrue PCP , 29, 30.true PCP . HoweverThe cut-off value of the Wako-BDG assay for the diagnosis of non-HIV PCP was 8.5 pg/mL in our study. This value was lower than the classical cut-off value of 31.1 pg/mL in a previous study including both HIV and non-HIV PCP patients. We believe that clinicians should be aware of the low BDG cut-off value for potential use in the diagnosis and management of patients with non-HIV PCP. Further multicenter prospective studies with standardized protocols are needed to confirm our results.Additional file 1: Fig. S1. Box plots of the beta-D-glucan levels in the PCP group (left) and the non-PCP group (right). Fig. S2. ROC curve for BDG by background disease. The cut-off value for BDG is 10.5 pg/mL with sensitivity and specificity of 89% and 92%, respectively in hematological malignancy (A) 8.0 pg/mL with sensitivity and specificity of 78% and 83%, respectively, in solid tumor (B) 16.5 pg/mL with sensitivity and specificity of 65% and 82%, respectively, in autoimmune disorder (C) 13.5 pg/mL with sensitivity and specificity of 67% and 80%, respectively, in idiopathic interstitial pneumonia (D).